STRUCTURAL-BASED MUTAGENESIS STUDY ON THE LOOP REGIONS OF STAR PROTEIN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 类固醇生成急性调节蛋白（星星）促进胆固醇快速移动到肾上腺和性腺线粒体，胆固醇成为合成所有类固醇激素的第一步的底物。 星星制造出应激反应和繁殖所需的线粒体膜。 星星的C-末端在脂质体存在下对蛋白水解具有抗性，这意味着该区域与膜相关。 与星星密切相关的两种蛋白质MLN 64和StARD 4的晶体结构是可用的，揭示了两个环与C-末端相邻。 环的构象变化可以使C末端螺旋旋转并打开屋顶，使胆固醇与蛋白质结合。 两个环含有带负电荷的残基，而C-末端富含带正电荷的残基，表明盐桥和氢键可以稳定结构。 环周围的表面的疏水性表明，该环可以是膜对接位点的脂质装载和卸载。 我们计划确定环区域中的带负电荷的残基是否在底物结合和膜结合中发挥作用。 首先，使用计算机图形实验室开发的“嵌合体”程序，利用密切相关的蛋白MLN 64和StARD 4的晶体结构构建人类星星蛋白模型。 从该模型中，我们将测量来自环区域的带负电荷的残基和来自C-末端的带正电荷的残基之间的侧链距离。 然后，我们将在计算机上突变这些残基，并评估它们对蛋白质几何形状和构象的影响。 潜在的启发式突变体，然后将建立和研究在体内的工作台。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The steroidogenic acute regulatory protein (StAR) facilitates the rapid movement of cholesterol into adrenal and gonadal mitochondria, where cholesterol becomes the substrate for the first step in the synthesis of all steroid hormones. StAR works out the mitochondrial membrane required for the stress responses and for reproduction. The C-terminus of StAR is resistant to proteolysis in ther presence of liposomes, implying that this region is associated with the membrane. Crystal structures are available for two proteins closely related to StAR, MLN64 and StARD4, revealing that two loops are adjacent to the C-terminus. Conformational changes of the loops could pivot the C-terminal helix and open the roof to allow cholesterol to bind to the protein. The two loops contain negative charged residues, while the C-terminus is rich is positively charged residues, suggesting that salt bridges and hydrogen bonds may stabilize the structure. The hydrophobicity of the surface surrounding the loops suggests that the loop could be the membrane docking site for lipid loading and unloading. We plan to determine whether the negatively charged residues in the loop regions play a role in substrate binding and membrane association. First, are building a human StAR protein model using the crystal structures of the closely related proteins MLN64 and StARD4, using the "Chimera" program developed in the Computer Graphics Lab. From the model, we will measure the side-chain distances between the negatively charged residues from the loop regions and the positively charged residues from C-terminus. We will then mutate these residue(s) in silico and evaluate their effects on the protein geometry and conformation. Potentially heuristic mutants will then be built and studied in vivo at the bench.